Historically, antimicrobial therapy dramatically reduced the mortality of Staphylococcus aureus infections. Because of multidrug resistance, successful treatment of S. aureus can be difficult to achieve. Novel therapeutic interventions are desperately needed. One promising approach is to develop drugs that target the regulators of virulence factor (VF) expression. A detailed molecular mechanism of action will greatly benefit the search for an appropriate target for regulator-specific drugs. VF regulation in S. aureus is controlled by the cooperative and redundant action of the products of many loci including a family of at least six MarR-family transcriptional regulators, the SarA-homologues. This proposal focuses elucidating the molecular mechanism of two SarA-homologues, Rot and SarU. Genetic evidence suggests that Rot and SarU have a reciprocal effect on the expression of VFs. Using alpha-toxin as an example we will define the molecular interactions that lead to Rot acting as a repressor of the gene encoding alpha-toxin (hlalpha) and SarU acting as an activator of hla transcription. Our hypothesis is that Rot is a constitutively expressed repressor that directly downregulates transcription of hlalpha. This repression is relieved by RNAIII, a riboregulator that is upregulated by both a direct interaction between SarU and the agr promoters and an indirect interaction of SarU on the sarA promoters. To test this hypothesis, we propose: Aim 1. To characterize mechanism(s) of Rot repression of a-toxin production that is antagonized by RNAIII. Aim 2. To characterize mechanism(s) of SarU activation of a-toxin production by demonstrating direct and temporally appropriate interactions between SarU and its target genes.